ANTIGEN RECOGNITION BY IGG4 ANTIBODIES IN HUMAN TRICHINELLOSIS

(1)

ANTIGEN RECOGNITION BY IGG4 ANTIBODIES IN HUMAN TRICHINELLOSIS

PINELLI E.*, VAN DER LUGT G.*, HOMAN W.**, VAN DER GIESSEN J.*** & KORTBEEK L.M.*

S u m m a r y :

The antibody isotype response to Trichinella spiralis

excretory/secretory (ES) products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 4 5 kDa component was strongly recognized by different antibody classes and subclasses. W e observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

K E Y W O R D S : human trichinellosis, ES antigen, 4 5 kDa, l g G 4 .

M

onitoring programs for detection o f trichinellosis in slaughterhouse animals have drama- tically decreased the incidence of human trichinellosis. However, in many parts o f the world, including Europe, it continues to occur. Recent out- breaks o f trichinellosis have b e e n reported from Ger- many, France, Spain and Italy (Ancelle et al, 1998;

Rodriguez et al., 1 9 9 9 ) ; (Pozio 1998).

In our laboratory the immunodiagnosis o f trichinellosis is performed by using an enzyme-linked immunosorbent assay (ELISA) in which the excretory/secretory (ES) products o f Trichinella spiralis muscle larvae are used as antigen (van Knapen et al., 1982). Analysis of this ES antigen by SDS-PAGE revealed that this antigen comprises a complex mixture. T h e use o f this antigen in an ELISA may give rise to false-positive results due to the presence o f antigenic components shared with

* Diagnostic Laboratory for Infectious Diseases and Perinatal Scree- ning, National Institute of Public Health and the Environment (RIVM), Bilthaven, The Netherlands.

**Research Laboratory for Infectious Diseases, National Institute of Public Health and the Environment (RIVM), Bilthaven, The Nether- lands.

***Microbiological Laboratory for Health Protection, National Insti- tute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Correspondence : Dr. Elena Pineli. Dept. of Parasitology and Myco- logy, LIS, National Institute of Public Health and the Environment (RIVM), P.O. Box 1. 3720 BA Bilthoven, The Netherlands.

Tel. : 31-30-2744277 - Fax: 31-30-2744418 E-mail : elena.pinelli@rivm.nl

other helminths. Therefore, to confirm results from the ELISA w e introduced the Western blotting technique.

Identification o f parasite antigens recognized by different antibody classes and subclasses can b e a valuable tool for the immunodiagnosis o f helminth infections (Magnaval et al, 1 9 9 1 ; G a d e a et al, 1 9 9 9 ) . W e observed that the IgG4 response directed to a 45 kDa component o f ES products of muscle larva was Tri-

chinella-specific. The measurement of this response can b e used for the immunodiagnosis o f trichinellosis.

MATERIALS AND METHODS

T

he excretory/secretory (ES) antigen was pre- pared using viable muscle larva of T. spiralis as previously described by Gamble ( 1 9 8 5 ) . T h e 45 kDa antigenic component was purified from freshly obtained muscle larva using two-step affinity chroma- tography as described by Homan et al. (1992)

SERUM SAMPLES

Serum samples from trichinellosis patients recently received at our laboratory and from an outbreak o f trichinellosis that took place in Slupsk, Poland in 1991 were used in this study. The patients here studied were all at an early stage of trichinellosis. All serum samples used tested positive for IgM and/or IgG by means o f ELISA using T. spiralis ES antigen. Infection w a s confirmed for several patients from the outbreak in Poland and for the other patients used in this study by identification o f Trichinella larva in muscle biopsy.

The source for T. spiralis infection for the outbreak was undercooked pork. Sera from our laboratory from echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis patients were also included.

W E S T E R N BLOTTING PROCEDURE

T h e ES antigen or the 45 kDa protein was solubilized under reducing conditions and electrephorized on

Parasite, 2001, 8, S168-S171 ANTIGENS

S168.

Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/200108s2168

(2)

HUMAN IMMUNE RESPONSE

10 % polyacrylamide gels in SDS according to Laemmli (1970). For Western blot analysis, antigens were electroforetically transferred onto nitrocellulose membrane according to the procedure o f Towbin et al. (1979).

Blots were incubated at 4°C overnight in 0.05 % Tween 20/phosphate-buffered saline (PBS/Tween) containing 1 % low fat milk (Protifar, Nutricia). Immunodetection was performed by incubation of the blots with serum samples diluted in 0.05 % PBS/Tween containing 1 % low fat milk. After sequential washing with PBS/Tween the blot was incubated with peroxidase conjugated monoclonal antibodies directed to the various IgG subclasses (CLB, T h e Netherlands) or with peroxidase conjugated goat anti-human IgM and IgG (Sigma).

Conjugated antibodies were diluted in PBS/Tween c o n t a i n i n g 1 % l o w fat m i l k . After w a s h e s in PBS/Tween, tetramethyl benzidine/di-octyl-natrium- sulpho-succinate (TMB/DONS, 0.06 % and 0.2 %, res- pectively in Dimethyl sulfoxide, DMSO) was added as substrate.

RESULTS

W E S T E R N BLOT ANALYSIS O F IG M AND IGG REACTIVITY T O ES ANTIGEN

A

nalysis of Western blots using T. spiralis-ES antigen indicates a specific banding pattern represented by bands of 125 kDa, 90 kDa, 78 kDa, 38 kDa, 31 kDa and 22 kDa that are recognized by IgM and IgG antibodies present in sera from a Tri- chinella-infected patient (Fig. 1). Bands of 45 kDa, 55 kDa and 58 kDa recognized by IgM and total IgG antibodies of trichinellosis patients appeared occasio- nally using sera from patients with other helminth infections. W e observed however, that IgM and total IgG antibodies present in sera from T. spiralis-infected patients reacted strongly to a 45 kDa antigenic component. T h e IgG response to this component was predominantly of the IgG1 and IgG2 subclass with a minor IgG3 response. Interestingly, a clear 45 kDa-specific IgG4 response was detected (Fig. 2). Therefore, we became interested to investigate further the parasite-specific IgG4 response of patients with trichinellosis.

FIG 1. - Western blot revealing bands recognized by Trichinella-spu- cific IgM and IgG antibodies. The ES antigen electrephorized on 10

% polyacrylamide gels in SDS was electroforetically transferred to nitrocellulose membrane. Indicated by arrows are the specific bands with molecular weight (Mol. Wt.) of 125 kDa, 90 kDa. 78 kDa, 38 kDa, 31 kDa and 22 kDa recognized by IgM (lane 1 to 4) and total IgG (lane 5 to 8) in serum from a trichinellosis patient from the outbreak that took place in Poland in 1991 (lane 1 and 5). Also indicated by lines are bands of Mol. Wt. 45 kDa. 50 kDa and 55 kDa which are recognized by IgM and IgG in sera of trichinellosis patients but are also ocassionally recognized by antibodies from other helminth infected patients (lane 4); echinococcosis (lanes 2 and 6), filariasis (lane 3 and 7), cysticercosis (lane 4 and 8).

FIG 2. - Reactivity of IgG subclass antibodies to 45 kDa component of ES antigen. A partially purified fraction of the component of ES antigen with a molecular weight (Mol. Wt.) of 45 kDa was electro- phorized on 10 % polyacrylamide gels in SDS was electroforetically transferred to nitrocellulose membrane. Indicated are results using scrum from a trichinellosis patient from the outbreak that took place in Poland in 1991; IgGl (lane 1), IgG2 (lane 2), IgG3 (lane 3) and IgG4 (lane 4).

Parasite, 2001, 8, S168-S171

X^{t h} ICT. Auqust

2000

^S169

(3)

PINEIXI E., VAN DER LUGT G., HOMAN W., VAN DER GIESSEN J., KORTBEEK L.M.

RECOGNITION O F E S ANTIGEN B Y I G G 4 ANTIBODIES

Fig. 3 shows the recognition of the ES antigen by IgG4 antibodies present in serum samples of trichinellosis patients. T h e intensity of this response varied from patient to patient and it was detected only when using sera from trichinellosis patients and not from patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis.

RECOGNITION O F 4 5 K D A COMPONENT O F E S B Y I G G 4 ANTIBODIES

In Fig. 4, the recognition of the 45 kDa component of the ES antigen by IgG4 antibodies is shown. The intensity of this response varied from patient to patient and it was observed only when using sera from trichinel-

losis patients. In contrast, no reactivity was found using sera from echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis patients. The serum samples used here were the same and in the same order as that described for Fig. 3. Noteworthy is the weak reactivity of IgG4 from patients shown in lane 15 and 16.

However, no reactivity at all was observed using the ES antigen and this same serum samples (Fig. 3).

DISCUSSION

I

nfection with Trichinella spiralis evokes a humoral immune response, predominantly of the IgM, IgG and IgA classes (van Knapen et al., 1982; Au et al., 1983; Feldmeier et al., 1987). In this study w e report

FIG 3- - Reactivity of IgG4 antibodies to ES antigen. The serum samples used included those of patients from the outbreak of trichinellosis taken place in Poland, 1991 ( 1 - 1 0 ) , and serum samples received at our laboratory of patients with trichinellosis (11-15), echinococcosis (16-17), filariasis (18- 19), cysticercosis (20-22), ascariasis (23- 25), strongyloidiasis (26-28) or toxocariasis (29-32).

Fig 4. - Reactivity of IgG4 antibodies to 45 kDa component of ES antigen.

The serum samples used included those of patients from the outbreak of trichinellosis taken place in Poland, 1991 ( 1 - 1 0 ) , and serum samples received at our laboratory of patients with trichinellosis (11-15), echinococcosis (16-17), filariasis (18-19), cysticercosis (20-22), ascariasis (23-25), strongyloidiasis (26-28) or toxocariasis (29-32). The serum samples used here were the same and in the same order as that described in Fig. 3.

S170 X^th ICT. Auqust

2000

Parasite, 2001, 8, S168-S171

(4)

HUMAN IMMUNE RESPONS

on the use o f Western blots for the identification of component o f the ES antigen recognized by IgM and IgG subclasses present in sera o f patients at an early stage of trichinellosis.

An interesting result from our study is the detectable IgG4 response directed to a 4 5 kDa component o f the ES antigen. This response was found to b e T. spiralis- specific since it was not detected using sera from patients with other helminth infections.

An increase in the production IgG4 during infection with helminths has been previously described (Ottesen et al., 1 9 8 5 ; M a g n u s s o n et al., 1 9 8 6 ) . H o w e v e r , increased IgG4 antibody levels has b e e n generally a s s o c i a t e d w i t h c h r o n i c a n t i g e n i c s t i m u l a t i o n (Magnusson et al., 1 9 8 6 ; Aalberse et al., 1983).

Detection of IgG4 levels in human trichinellosis has not been extensively studied. However, from an earlier follow-up study it was shown that a significant higher number of IgG4 Trichinella-positive sera were found by means of ELISA, during the chronic stage o f infection. T h e authors suggest that the IgG4 response can discriminate between an early and a late infection with T. spiralis (Ljungstrom et al., 1988). B y identifying the target antigens o f IgG4 and using a purified a fraction o f o n e of these antigens w e observed that using Western blots, this response could also b e detected during an early stage of trichinellosis. Taking together, monitoring the 45 kDa-specific IgG4 response c a n b e a valuable tool for the immunodiagnosis o f human trichinellosis.

AALBERSE R . C . , VAN DER G.R. & VAN LEEUWEN J . Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4-restricted response.J. Immunol., 1983, 130, 722-726.

ANCELLE T . , DUPOUY CAMET J., DESENCLOS J . C . , MAILLOT E., SAVAGE HOUZE S., CHARLET F., DRUCKER J . & MOREN A. A mul-

tifocal outbreak of trichinellosis linked to horse meat imported from North America to France in 1993- Am.

J. Trop. Med. Hyg., 1998, 59, 615-619.

Au A.C., Ko R . C . , SIMON J . W . , RIDELL N.J., WONG F.W. & TEM-

PLER M.J. Study of Acute Trichinosis in Ghurkas : Specificity and sensitivity of enzyme-linked immunosorbent assays for IgM and IgE antibodies to Trichinella Larval antigens in dia- gnosis. Trans. R. Soc. Trop. Med. Hyg., 1983, 77, 412-415.

FELDMEIER H., FISCHER H. & BLAUMEISER G. Kinetics of humoral response during the acute and the convalescent phase of human trichinosis. Zentralbl. Bakteriol. Mikrobiol. Hyg., A., 1987, 264, 221-234.

GADEA I., AYALA G., DIAGO M.T., CUNAT A. & GARCIA DE LOMAS

J. Immunological diagnosis of human cystic echinococ- Parasite, 2001, 8, S168-S171

cosis : utility of discriminant analysis applied to the enzyme-linked immunoelectrotransfer blot. Clin. Diagn.

Lab. Immunol., 1999, 6, 504-508.

GAMBLE H.R. Trichinella spiralis: Immunization of mice using monoclonal antibody affinity-isolated antigens. Exp.Para- sitol., 1985, 59, 398-404.

HOMAN W.L., DERKSEN A.C & VAN KNAPEN F. Identification of

diagnostic antigens from Trichinella spiralis. Parasitol.

Res., 1992, 78, 112-119.

LAEMMLI U . K . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227, 680-685.

LJUNGSTROM I., HAMMARSTROM L., KOCIECKA W. & SMITH C.I. The

sequential appearance of IgG subclasses and IgE during the course of Trichinella spiralis Infection. Clin. Exp.

Immunol, 1988, 74, 230-235.

MAGNAVAL J . F . , FABRE R., MAURIERES P., CHARLET J.P. & DE LAR-

RARD B . Application of the Western blotting procedure for the immunodiagnosis of human toxocariasis. Parasitol.

Res., 1991, 77, 697-702.

MAGNUSSON C.G., CESBRON J . Y . , DJURIUP R., CAPRON A. &

JOHANSSON S.G. Raised serum IgG4 levels in patients with atopy and filariasis : Application of an automated particle- counting immunoassay using monoclonal antibody. Int.

Arch. Allergy Appl. Immunol, 1986. 81, 238-244.

OTTESEN E.A., SKVARIL F., TRIPATHY S.P., POINDEXTER R.W. & Hus-

SAIN R. Prominence of IgG4 in the IgG antibody response to human filariasis. J. Immunol, 1985, 134, 2707-2712.

POZIO E. Trichinellosis in the European union: Epidemiology, ecology and economic impact. Parasitol. Today, 1998, 14, 35-38.

RODRIGUEZ O.M., ABAD J.M., DE HARO T., VILLA R.R. & GOMEZ

G., V Human Trichinellosis in southern Spain : Serologic and epidemiologic Study. A.J. Trop. Med. Hyg., 1999, 61, 834-837.

TOWBIN H , STAEHELIN T. & GORDON J . Electrophoretic Transfer

of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad.

Sci. USA, 1979, 76, 4350-4354.

VAN KNAPEN F., FRANCHIMONT J.H., VERDONK A.R., STUMPF J .

& UNDEUTSCH K. Detection of specific immunoglobulins (IgG, IgM, IgA, IgE) and total IgE Levels in human trichinosis by means of the enzyme- linked immunosorbent assay (ELISA). Am. J. Trop. Med. Hyg., 1982, 31,

ANTIGEN RECOGNITION BY IGG4 ANTIBODIES IN HUMAN TRICHINELLOSIS